Fungi are microscopic, spore-bearing organisms that lack chlorophyll and therefore derive nourishment from dead or living organic matter. Introductory Mycology (eds.). Alexopoulos, C. J., Mims, C. W., and Blackwell, M. (1996). 4th edition. Chapter 1. Because they share characteristics of both plants and animals, they are classified separately in the kingdom Fungi. Within this kingdom, there are the “filamentous fungi”, so named because their vegetative bodies consist of small thread-like filaments referred to as “hyphae”. Typically, the hyphae grow in a branching fashion, spreading over or within the substrate used as a source of nourishment, thereby forming a network of hyphae called “mycelium”. Thus, the mycelium is the vegetative body of the fungus. In the life cycle of most filamentous fungi, the vegetative mycelium gives rise to either asexual or sexual spores. Asexual spores are referred to by a variety of names, but commonly used terms are “conidia”, “condiospores”, or simply “spores”. The vegetative mycelium of the fungus may differentiate, with the appropriate biological and environmental cues, into a sexual reproductive spore-bearing structure. Some fungi produce sizable, fibrous (“fleshy”), spore-bearing reproductive structures variously called “mushrooms” “fruit bodies” “basidiocarps”, “ascocarps”, “conks”, or “basidomes”. The fruit bodies of some fungi are edible; being valued for their culinary, nutritional, or medicinal qualities, and, as such, are highly sought after or grown commercially.
The fruit body may be differentiated into specialized tissues such as the fleshy umbrella-shaped cap (pileus), stem (stipe), cup at the base of the stem (volva), and gills (lamallae) bearing the sexual spores. A thin tissue known as the veil (velium) may cover the underside of the cap. The veil ruptures as the fruit body approaches maturity, exposing the gills and permitting the discharge of the sexual spores into the environment. However, the fruit bodies of some fungi lack gills all together, and instead are composed of fleshy tissue perforated with small pores or locules bearing the sexual spores. Sexual spores produced by the fleshy reproductive structures of fungi are described by numerous terms, as for example, “ascospores”, “basidiospores”, or simply “spores”.
Thus, the fruit body of fungi is functionally comparable to the reproductive structure of plants known as the flower, whereas both asexual and sexual spores are comparable to the seed of plants, being important in the dispersal and survival of the fungus in nature. Under suitable environmental conditions, the spore germinates to form another generation of vegetative hyphae and so completing the life cycle of the fungus.
Filamentous fungi have a vital role as one of the primary decomposers within their varied natural habitats. They also have a large impact on food production. Some fungi, such as mushrooms, are used as food, while others are plant pathogens that are responsible for devastating crop losses all over the world. Filamentous fungi are also important in industry and medicine as they secret a diverse array of enzymes (e.g. proteases, lipases) as well as primary (e.g. organic acids) and secondary metabolites (e.g. antibiotics penicillin and cephalosporin). The cultivated mushroom Agaricus bisporus is a significant crop, with a world-wide production in 1990 of 1.5 million tons. Filamentous fungi are also attractive as hosts for large-scale production of both homologous and heterologous proteins, because they have the capacity to secrete substantial amounts of proteins.
About 40% of the commercially available enzymes are derived from filamentous fungi. Lowe, Handbook of Applied Mycology. Fungal Biotechnology (eds.) Arora, D. K. Elander, R. P. & Mukerji, K. G. 681-708 (Marcel Dekker, New York; 1992). These enzymes are usually produced by species of the genera Aspergillus and Trichoderma. Because they secrete large amounts of protein into the medium, they can be grown in large-scale fermentation, and they are generally accepted as safe for the food industry.
General problems associated with the commercial cultivation of mushrooms (A. bisporus) include diseases caused by pathogens like Verticillium fungicola (dry bubble), Trichoderma harzianum biotypes 2 and 4 (green mold), Pseudomonas tolaasii (blotch), and dsRNA viruses (La France disease and MVX), the major insect pest [sciarid fly (Lycoriella mali)], an extremely short shelf life of the product related to bacterial spoilage and rapid senescence, and browning (bruising) of the fruit body associated with the action of endogenous poly-phenoloxidases (PPO, like tyrosinase). To further improve product quality, conventional breeding programs for A. bisporus have been only moderately successful and may not be sufficient in the long term. This is because conventional breeding techniques for fungi are highly time consuming, and because the genetic variation in commercially available strains is limited, offering little advancement by selection (Horgen et al. “Homology between mitochondrial DNA of Agaricus bisporus and an internal portion of a linear mitochondrial plasmid of Agaricus bitorquis” Curr Genet. 1991 June; 19:495-502.
In the case of A. bisporus, the main problem for effective breeding strategies is caused by the rather abnormal life-cycle involving the unusual simultaneous segregation of either parental nucleus into one basidiospore. After outgrowth of this basidiospore, heterokaryotic mycelium is formed containing nuclei and genetic characteristics that do not differ from those present in the parental mycelium. In addition, only little recombinational activity is observed during meiosis (Summerbell et al. 1989. Genetics 123: 293-300).
For this reason, investigators all over the world have attempted to develop a transformation system for commercial mushrooms, such as A. bisporus, for the introduction of novel characteristics. For other fungi, as well as plants, animals, and bacteria, the application of gene transfer technology is quite common and has already resulted in commercial application. However, the absence of an efficient, reproducible, stable transformation system generally applicable in a wild-type background in many fungi has strongly hampered molecular-biological research on such organisms.
Current transformation techniques for fungi have included a combination of CaCl2 and polyethylene glycol (PEG), electroporation, and particle bombardment to introduce DNA into protoplasts, mycelium, or spores. These have been either without success, or not reproducible. The lack of a practical gene transfer system is the single largest obstacle precluding the use of molecular approaches for the genetic improvement of mushrooms. Despite considerable interest in the development of a transformation scheme, no method is in general use today, due to low efficiency or lack of utility and convenience.
In recent approaches, several fungi, including A. bisporus, have been transformed using an Agrobacterium-based transformation system. Although these methods are more convenient than the existing protoplast-based schemes, they have thus far suffered from a comparably low efficiency of transformation using complicated systems.
For example, Gouka et. al. describe a transformation procedure for targeted homologous recombinations in fungi, (Gouka et. al. Nature Biotechnology Vol 17 Jun. 1999, “Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination” pp 598-601). According to this procedure a specifically engineered A. awamori recipient strain containing a 3′-deleted nonfunctional pyrG gene and an Agrobacterium strain containing a binary vector suitable for restoring the pyrG gene by recombination are used. Homologous recombination between the repair construct and the recipient host result in restoration of functional pyrG gene and integration of the vector at the pyrG locus. The paper reported a high of 150 transformants per 107 conidia.
De Groot et. al report yet another Agrobacterium-based method of transforming filamentous fungi, (De Groot et. el. Nature Biotechnology Vol 16 Sep. 1998, “Agrobacterium tumefaciens-mediated transformation of filamentous fungi” pp. 839-842). This paper investigated the ability of Agrobacterium to transfer T-DNA to the A. awamori protoplasts (vegetative cells with the cell walls removed) and conidia. The transformation frequency varied from approximately 300 to 7200 transformants per 107 protoplasts, which was up to 600 times higher than PEG transformation rates. When conidia were used, the transformation frequency varied from 1000 to 9000 transformants per 107 conidia. Vegetative mycelial tissue was also used.
Other fungi transformation schemes are disclosed in WO95/02691 and WO98/45455. All of these have focused on transformation using protoplasts, spores, and vegetative mycelium as the recipient tissue.
Numerous scientists are using the Penn State genetic transformation methodology in their research efforts. There are a growing number of examples in the literature where the methodology has enabled the expression of recombinant proteins and RNA transcripts (i.e. hairpin RNA-induced RNAi) in A. bisporus. In each case, a traditional approach was taken in which the transgene construct entailing a nucleotide sequence-of-interest joined to an operable promoter and terminator was stably integrated in the Agaricus genome, resulting in a fruiting body having both a transgenic genotype and phenotype. An A. bisporus line generated in this manner currently has low commercial value, as genetically modified mushrooms are currently not preferred in the marketplace. Further, if the objective is to utilize A. bisporus as a manufacturing platform, a traditional approach to genetic modification often fails to achieve high-level expression of the protein-of-interest. Therefore, there is a continual quest to identify new strategies for attaining higher levels of protein production.
As can be seen from the foregoing, there is a continuing need in the art for development of effective, convenient, and expeditious fungal transgenic protocols.
It is thus an object of the present invention to provide a transgenic manipulation system for fungi that will accomplish the foregoing need.
A further object of this invention is to provide mechanisms for application of transgenic techniques to increase yield, disease, and pest resistance, product quality, shelf life, or culinary, nutritional, or medicinal value, to produce commercially, or other such protocols.
It is yet another object of the invention to provide polynucleotide constructs, vectors, transformed cells for use in such transgenic manipulation protocols.
Other objects of the invention will become apparent from the description of the invention that follows.